X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. The selective pressure for B cells expressing normal Btk suggests that introduction of the Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. Using retroviral vectors optimized for expression in HSC and transplantation of transduced stem cells into Btk/Tec-/- recipients we have achieved: sustained marking and B lineage engraftment, and selective expansion of Btk expressing B lineage cells. This has led to full restoration of both the pre-B and transitional B cell development transitions, and of Bkt dependent functions including B cell receptor signaling, T-independent immune responses, and circulating immunoglobulin. In the current proposal we will refine this viral delivery system to optimize its clinical feasibility for rescue of Btk function. We will test the hypotheses that: 1.) The selective outgrowth of the corrected cells will be sufficient to restore Btk dependent functions in a minimally or non-myeloablated setting; 2) The rescue of B cell development and function will protect gene corrected animals from life-threatening bacteremia; 3) Self-inactivating retroviral vectors containing unique B-lineage enhancer/promoter elements will mediate sustained, temporally appropriate Btk expression and rescue function in vivo and in vitro; 4) Incorporation of the insulator elements into these SIN vectors will promote more consistent expression and, more importantly, reduce the risk of viral enhancer mediated mutagenesis by limiting activation of host gene expression; 5) These viral vectors will rescue the deficient generation of surface lg+ B cells from transduced XLA HSC in vitro and in vivo. Recent clinical trials in immunodeficiency disorders highlight the great potential for genetic correction as well as an unanticipated risk for development of onco-retroviral toxicity. While conceptually simple, implementation of definitive genetic therapy in XLA requires a concerted program of basic and applied research aimed at developing safe and efficient vector systems capable of appropriate, specific and sustained B lineage gene expression. Overcoming these technical challenges will likely provide important insight into therapies for other more common genetic disorders lacking a selective advantage for corrected cells.